The present invention relates to the field of the cultivation of cells, especially of adherent tissue cells such as liver cells. More in particular, the invention relates to the field of biological methods and reactors for the cultivation and/or maintenance of cells, especially liver cells, and to the use of such methods in a bio-artificial liver system (BAL).
It is generally known that most tissue cells require a solid support on which to grow and divide.
Although it is possible to culture adherent tissue cells in ordinary vessels, such as glass bottles or Petri dishes, during which the cells adhere to the wall of the vessel, usually special reaction vessels or bottles with a high surface area are used so as to provide increased capacity for cell attachment. One way to improve said surface area is to use a solid support for cell adherence. Such solid supports are known in the art; examples include glass beads, microcarriers and cellulose fibers.
A special problem in the cultivation of adherent cellsxe2x80x94compared to the cultivation of cells in suspension or in confluent layersxe2x80x94is to provide sufficient nutrients and/or oxygen to the cells and/or provide for sufficient removal of waste products and/or carbon dioxide. This is especially a problem with cells that put stringent demands on both oxygenation as well as the removal of waste products, such as liver cells.
The non-availability of suitable solid supports and methods for the in vitro cultivation of liver cells has over the last 40 years severely hindered the development of the so-called bio-artificial liver (BAL) systems, systems that could be used in patients with liver defects for the support and/or replacement of the natural liver function.
As acute liver failure has a very poor prognosis and is usually fatal to the patient within days or even hours [see for instance Devlin et al., Hepatology Vol. 21, No. 4 (1995), pages 1018-1024 and Lake and Sussman, Hepatology, Vol. 21, No. 3 (1995), pages 879-882, describing the general problems in the art of the treatment of liver failure, both incorporated herein by reference], because livers for transplant are not readily available, a BAL system that could support and/or replace liver function, for instance during the time the patient awaits for a liver to become available for transplant and/or to bridge the period until the liver of the patient sufficiently recovers and/or regenerates by itself and/or as a result of treatment, would be highly desirable.
However, due to the abovementioned lack of suitable methods and/or materials for cultivating and/or maintaining liver cells in vitro, the bio-artificial liver systems from the prior art have so far proved insufficient, because they do not fully replace all the functions carried out by the liver of the patient in vivo, because they have insufficient capacity, and/or because the time during which they are therapeutically effective is too limited for practical use.
The history of bio-artificial liver systems has been described in a number of recent articles, notably Nyberg et al., the American Journal of Surgery, Vol. 166, November 1993, p. 512-521, and Sussman and Kelly, Scientific American, May-June 1995, p. 59-77, incorporated herein by reference.
As described in these articles, the earliest liver support systems were based on hemodialysis, charcoal hemoperfusion, or cross-hemodialysis either between humans or between humans and animals. Also, extra-corporeal liver perfusion has been tried.
All these systems have been found to be insufficient. As stated by Nyberg et al.:
based on the limited success achieved by early liver support techniques, the concept evolved that liver functions essential for survival would be best provided by mammalian liver preparations that allowed sustained or repetitive application. These liver preparations, commonly referred to as hybrid or bio-artificial systems, contain biological components within a synthetic framework. Biological components may include isolated liver enzymes, cellular components, slides of liver or cultured hepatocytes. Hepatocytes may be implanted in the patient or perfused extra-corporally. Hepatocytes systems have shown the greatest promise for bio-artificial liver support. When compared with cellular component and isolated enzyme systems, hepatocyte systems should supply a greater number of liver functions, since they utilize intact, metabolically active liver cells ( . . . ). One major advantage of the hepatocyte bio-artificial liver over traditional hepatocyte transplantation and earlier support techniques, such as cross-circulation and extra-corporal liver perfusion, is that the bio-artificial liver can be constructed from semipermeable materials that provide a barrier between the hepatocytes and the host immune system. As a result bio-artificial liver therapy may be performed without immunosuppression, and hepatocytes from different species (xenocytes) may be used within the bio-artificial liver.
The disadvantages of bio-artificial liver systems include ( . . . ) the problem of maintaining normal hepatocyte viability and function at the high cell density necessary for clinical application. For example, when hepatocytes are grown on a plastic surface with standard cell culture medium, they lose their gap junctions in about 12 to 24 hours; they also flatten and become a granular; tissue specific functions are lost in 3 to 5 days, followed by hepatocyte death within 1 to 2 weeks. As a result, improved techniques of cell culture have become necessary for the application of bio-artificial liver support systems.
A number of different approaches to the cultivation of hepatocytes and related cells for use in or as BAL-systems have been described. However, the prior art hepatocyte systems also suffer from problems with regard to capacity and effective working time, see Sussman and Kelly:
With regard to the provision of sufficient metabolic capacity, it is not clear exactly how much liver necrosis is fatal. Animal experiments suggest that at least 30% of the liver""s original function must be preserved in order to survive. The adult human liver contains approximately 1000 gm of hepatocytes, which are the metabolically active cells. Thus we have proposed that effective liver assistance will require the equivalent of 300 to 400 gm of cells. Two sources of hepatocytes are available: freshly isolated cells (primary cultures) and cells grown in continuous culture (cloned or immortalized cells). Cells that have been isolated from a normal human or animal liver retain many of their functions ( . . . ) the technology has severe limitations.
Artificial livers that use freshly isolated cells have so far provided only a fraction of the necessary metabolic capacity. Hepatocytes do not divide after they have been isolated, so a steady supply of new cells is required. Coupled with the labour-intensive nature of cell preparation, this makes it almost impossible to scale up production to meet current needs in a cost-effective manner. Moreover, freshly isolated cells do not appear to last very long during treatment. A liver assist device that lasts for only 6 to 7 hours, as some have been reported to do, clearly falls short of allowing liver regeneration. Finally, production of any such device using animal cells entails a number of problems, especially in areas of sterility and lot-to-lot variability.
Uchino et al., ASIAO Transactions 1988;23;972-977 describe a hybrid bio-artificial liver composed of multiplated hepatocyte monolayers. A total of 80 grams of cultured adult dog hepatocytes was cultured in a reactor comprising a stack of 200 collagen coated borosilicated glass plates. These hepatocytes were viable and functioned well during 4 weeks in perfusion culture. This bio-artificial liver was tested in anhepatic dogs. The longest survival obtained was 65 hours.
However, a serious drawback of this system, besides the complexity of constructing and using a 200 glass plate-reactor, is that the monolayer culture of hepatocytes on said plates precludes the advantageous formation of hepatocyte aggregates. It is well known in the art that hepatocytes cultured in or as aggregates function both longer and better than hepatocytes cultured in monolayers, showing higher activity and better differentiation.
Another approach in the development of bio-artificial liver systems has been the use of hollow fiber bioreactors in which liver cells are present in the extra fiber (extraluminal) space while a liquid medium is pumped through the fiber lumen (intraluminal space), usually by perfusion with whole blood or plasma.
Rozga, Demetriou et al., Biotechnology and Bio-engineering, Vol. 43 (1994), incorporated herein by reference, give an overview of the current hollow fiber systems. Their own system consists of a high flow plasma perfusion circuit comprising a charcoal column and a porous hollow fiber module with 5 to 6xc3x97109 microcarrier-attached porcine hepatocytes seeded into the extra fiber compartment. Because of the use of solid support (collagen coated dextran microcarriers), the surface area available for hepatocyte attachment is increased.
However, this design requires a separate membrane oxygenator for the oxygenation of the plasma to be incorporated into the perfusion circuit so as to provide sufficient oxygen to the hepatocytes in the hollow fiber module. Therefore, said oxygenation as well as the removal of carbon dioxide are dependent upon limiting factors such as the solubility of oxygen and carbon dioxide in the plasma and the transport of the oxygenated plasma throughout the reactor. Because of these limitations said hollow fiber reactor cannot easily be scaled up to a capacity required for practical therapeutic application.
Furthermore, this reactor is used with a very xe2x80x9cclosed pathxe2x80x9d column with a high density of the microcarriers, which leads to the formation of microcarrier pellets and to mass transfer problems with regard to the cells at the center of such a pellet.
Another disadvantage of this system is that the hepatocytes first have to be immobilized on the micro-carrier before the hepatocytes can be introduced into the hollow fiber reactor. This involves further complicating processing steps that can lead to loss of cell viability.
Sussman and Kelly, mentioned hereinabove, describe a hollow fiber-based bio-artificial liver system in which liver cells are attached to capillaries through which whole blood from the patient is pumped.
According to this system, the liver cells are oxygenated by the patient""s blood, becausexe2x80x94as stated by the authorsxe2x80x94xe2x80x9cplasma does not provide the oxygen carrying capacity of whole bloodxe2x80x9d.
Furthermore, perfusion with whole blood can lead to the fibers and/or the pores thereof within the bioreactor getting clogged, which problem could only be solved by totally replacing the hollow fiber module, requiring a fresh isolation/immobilization of the hepatocytes.
Other disadvantages of this and other hollow fiber systems using whole blood as the liquid medium are that xe2x80x9cthe hollow fiber membrane must first act as a plasma separator before any significant transport of nutrients and metabolites can take place across the fiber wallxe2x80x9d, and that it xe2x80x9crequires systemic anticoagulation with heparin to prevent clotting in the modulexe2x80x9d.
Also, in order to overcome problems with the isolation of cells, in this BAL-system a special cell line named C3A derived from a liver tumor of a child is used. However, with regard to activity and function, the use of such tumor-derived cell lines is generally less preferred in the art than the use of isolated primary hepatocytes, also from a safety standpoint.
Furthermore, the C3A cell line used by Sussman lacks some very important functions of primary hepatocytes. Also, the C3A cells are less differentiated, and therefore less active than primary liver cells.
A somewhat different hollow fiber system is described by Nyberg et al., mentioned hereinabove; hepatocytes are suspended in a collagen gel, which is injected into the lumen of hollow fibers. After that, the extra fiber space of the bioreactor is perfused with medium for 24 hours, after which the gel contracts within the fibers, thereby creating a third space which is perfused with medium.
The idea behind this three-compartment design is that blood can be pressed through the extra fiber compartment, whereas the gel entrapped cells are nourished and possibly stimulated by the factors present in the medium flowing through a path adjacent to the contracted collagen.
However, this system also requires a complicated and time consuming pre-immobilization of the hepatocytes.
Another BAL-system based on capillaries for hepatocyte immobilization is described by Gerlach et al., Transplantation, Vol. 58, No. 9 (1994). Their bioreactor consists of a three dimensional framework for decentralized cell perfusion with low metabolite gradients and decentralized oxygenation and CO2-removal, consisting of a woven network of four discrete capillary membrane systems, each serving different purposes, i.e., I, plasma inflow (polyamide fibers); II, oxygenation and carbon dioxide removal (hydrophobic polypropylene fibers or silicon fibers); III, plasma outflow (polysulfone fibers); and IV, sinusoidal endothelial co-culture (hydrophilic polypropylene fibers). These capillaries must be woven in such a way that the majority of hepatocytes find all four types of membranes in their surroundings.
This reactor was used with 2.5xc3x97109 pig hepatocytes with a viability between 88 and 96%, which were co-cultured with autologous sinusoidal endothelial cells present in the co-cultured compartment of the reactor.
In this type of hollow fiber bioreactor the liver cells have to be attached directly to the hollow fibers as no further matrix material for cell attachment is present in the reactor. In order to obtain sufficient attachment of the cells, the surfaces of the fibers must first be coated with a proteineous basement membrane product, such as Matrigel(copyright) or other collagen-based materials, requiring a separate and expensive pretreatment step. Even so, as hollow fibers are not specifically designed and/or suited for use as a solid support in cell cultivation, the attachment and the speed thereof permitted by and/or obtainable with said reactors is limited and heavy inoculum charges are required when seeding the reactor.
Furthermore, the average fiber distance within said three-dimensional fiber framework is about 500 xcexcm, leading to the formation of large cell aggregates of comparable size. Again, these large aggregates can lead to mass transfer problems with regard to the cells in the center of said aggregate.
Also, it is well known that hollow fibers are difficult to process, and in this respect the manufacture of the very complicated three-dimensional fiber network described by Gerlach et al., comprising four separate discrete capillary systems, suffers from a disadvantage from an economical point of view. Also, this reactor is complicated to operate, requiring multiple separated inlet/outlet control systems.
A general problem of all the abovementioned hollow fiber bioreactors of the prior art is that the liquid medium (blood, plasma) to be treated is separated from the hepatocytes by the hollow fiber membrane; in other words, that there is no direct contact between the liquid medium and the hepatocytes in the reactor. Nutrients and substances to be removed from the liquid medium and/or to be secreted into the liquid medium, have to pass through said membrane barrier in order to reach the hepatocytes and the liquid medium, respectively. The passage through the membrane can lead to transport phenomena that can limit the achievable mass transfer, and therefore the efficiency of the BAL-system.
Also, the membranes can get clogged, especially when perfusion with whole blood is used. In that case the BAL system or parts thereof have to be replaced, which means that therapy has to be interrupted or even stopped.
Another important limiting factor in the membrane transport is the molecular weight cut off of the membrane, see Nyberg et al.:
Permeability and membrane molecular weight cut off influence waste removal, product delivery, and immune activation. Performance of biotransformation functions and the removal of nitrogenous wastes are important functions of the bio-artificial liver, along with the removal of red blood cell breakdown products such as bilirubin. The production of coagulation proteins and other serum proteins by hepatocytes in the bio-artificial liver may also be beneficial to patients with liver failure. However, these proteins are of comparable sizes to antibodies, which could have an adverse effect when directed against nonautologous hepatocytes in the bioreactor. Alternatively, small peptide products of the hepatocytes may exit the bioreactor and serve as antigenic stimulant in the patient. Whether these foreign molecules will result in harmful cytokine production, immune complex formation, or serum sickness in patients with liver failure remains to be determined. Potential side-effects must be addressed experimentally in order to determine the best molecular weight cut off for use in the bio-artificial liver.
Clinical treatment of hepatic failure requires large scale, high density hepatocyte culturing. In many bioreactors this gives rise to the formation of non-physiological hepatocyte pellets. Hepatocytes in the center of these large aggregates show poor metabolic activity and even possible necrosis due to high gradients as a result of hindered transport of nutrients and oxygen to and carbon dioxide, toxins and cell products from these cells. This is in contrast to the in vivo liver where every hepatocyte is in close contact with the blood. Besides, in most systems substrate exchange depends on diffusion which further limits mass transfer compared to the in vivo situation where hepatocytes function under perfusion conditions with low gradients.
Also, the bioreactors of the prior art are limited with respect to the amount of liquid medium that can be withdrawn from the hollow fiber lumen, as in general the fusion transport will be too slow. Therefore, an active withdrawal of liquid medium from within the hollow fibers will be required, even so, the total flow through the hollow fiber membrane will be very slow and/or lead to the undesired formation of gradients, even with a high flow of liquid medium through the hollow fibers themselves.
Another general problem with the bio-artificial liver systems of the prior art is that they require the use of liver cell preparations with a high viability ( greater than 80%) and a high attachment. As already acknowledged by Sussman and Kelly hereinabove, the production of such cells is a very costly, complex and time-consuming process requiring isolation and subsequent cultivation of suitable liver cells in sufficient viability and quantity which involves complicated procedures that do not reliably afford the required results, even when carried out by qualified experts.
Furthermore, known hepatocyte-containing BAL-systems cannot be stored before use for a prolonged period of time because the viability and function of the liver cells in the reactor cannot be maintained at a therapeutically acceptable level.
Also, the only technique available for preserving isolated liver cells over a longer period of time, i.e., cryopreservation, does not afford cells that are suitable for use with known BAL-systems, see Rozga et al., mentioned hereinabove:
availability of cells on demand becomes a very important consideration in the clinical setting where treatment of patients with FHF is carried out emergently, on short notice and at all hours. However, [cryopreservation] may result in a significant loss of cell viability [. . . ] and attachment (as much as 50%). [. . . ] Therefore, in clinical settings, we prefer the use of freshly isolated, well attached hepatocytes.
Because of these problems, the known BAL-systems cannot be used as xe2x80x9coff the shelfxe2x80x9d units that can be kept and/or maintained in hospitals until their use is required, as is the case with other artificial systems for organ support such as for instance dialysis machines or artificial heart or lung systems. Also, replacement during therapy of a spent primary liver cell based BAL system of the prior art with insufficient function with a fresh BAL system is usually not economically feasible over a prolonged period of time.
For instance, Demetriou reports that after 6 hours of use, 50% of the primary liver cells within his reactor die, whereas within 24 hours all cells have died. Better results have been obtained by using immortalized cells or the C3A cell line reported by Sussman et al., however, the use of this hepatoblast derived cell line has other disadvantages as already mentioned hereinabove.
In view of the above, there is a continuing need for bio-artificial liver systems that do not have the abovementioned disadvantages of the prior art systems.
The British patent application 2,178,447 describes a matrix for cell cultivation in vitro providing an increased available effective surface area for cell attachment provided by a fiber network or open-pore foam with a suitable pore size 10 xcexcm to 100 xcexcm. This matrix material can be provided in the form of a sheet or mat or in the form of particles or flakes, in which latter form it is marketed by Bibby Sterilin under the name Fibra-Cel(copyright). As a sheet or mat, this matrix material has an appearance like filter paper or tissue paper, or thin porous felt.
This matrix material has some specific advantages over micro-capsules, which are costly and delicate to produce and give problems at high cell density growth, because frequently cells at the center of the capsule die. Also, the microcapsules may burst prematurely losing their contents and each new inoculation requires a fresh encapsulating procedure. Compared to microcarriers the matrix material according to GB-A-2,178,447 has the advantage that the cells are immobilized within the matrix structure. With microcarriers, these cells are immobilized on the outside of the carrier particles, making them susceptible to shear stress and particle collisions, for instance during preparation or packing of the reactor.
Furthermore, in the matrix material according to GB-A-2,178,447, the cells can proliferate along the fibers of the sheet in three dimensions (3D), rather than in two dimensions as in conventional tissue culture bottles, flasks or Petri dishes or on microcarrier beads or hollow fibers. Cells may attach themselves to more than one fiber and cell growth takes place in the internal volume of the fiber matrix. For these reasons, this and similar matrix materials are known in the art as xe2x80x9c3D-carrier matricesxe2x80x9d.
Another advantage of said 3D-matrix material is that it does not require the heavy inoculum charges of two dimensional systems (20-30% of the final amount of cells at saturation), but can be inoculated at amounts of less than 10%, and as low as 5%. The three dimensional network provides for a higherxe2x80x94and quickerxe2x80x94xe2x80x9ccapturexe2x80x9d of the cells, thereby also making it possible to use cells with sub-optimal attachment.
The GB-A-2,178,447 furthermore describes a number of potential bioreactor geometries employing the matrix material described therein. One of these comprises a sheet of said matrix material, rolled up into a spiral between two flattened tubes, wherein each alternate flattened tube serves a conduit, one for liquid nutrient medium, and the other for gases such as oxygen, air, Co2 and water vapor.
However, GB-A-2,178,447 is not directed to the construction of bio-artificial liver systems, nor to the specific problems relating to the cultivation and/or maintenance of hepatocytes therein. In particular, GB-A-2,178,447 does not relate to the special problem of supplying sufficient oxygen to highly oxygen dependent hepatocytes.
In fact, the use of the xe2x80x9cspiral woundxe2x80x9d reactor according to GB-A-2,178,447 for culturing and/or maintaining hepatocytes would in practice lead to insufficient oxygenation, because the oxygen is supplied by means of just one conduit covering the entire length of the matrix mat. The use of such a single conduit would lead to the generation of an undesired oxygen gradient along its length or even to local oxygen depletion, especially when the reactor is scaled up by increasing the number of matrix windings.
Also, the bioreactor construction according to GB-A-2,178,447 contemplates a separate conduit for the supply and/or removal of the liquid medium, so that during use as a BAL, nutrients, toxins and other substances to be absorbed or secreted would have to pass through the membrane surrounding said conduit in order to reach the hepatocytes, giving the problems with regard to membrane transport and mass transfer as described hereinabove.
Furthermore, the use of a singular spiral wound conduit for liquid transport can lead to an inhomogeneous supply of liquid medium to all the parts of the bioreactor, for instance by the generation of undesired gradients.
All these factors make the matrix material as such and the bioreactor according to GB-A-2,178,447 unsuited for use in the cultivation of liver cells and/or for use as a BAL.
It is therefore a first object of the invention to provide an improved solid support and bioreactor for the cultivation and/or maintenance of adherent cells, especially liver cells, with improved cell adherence properties and improved supply and/or removal of gaseous components such as oxygen and carbon dioxide, even when used in or as a large scale bioreactor.
It is a further object of the invention to provide an improved solid support and bioreactor enabling direct liquid contact between the cells and the liquid medium to be treated while at the same time maintaining a homogeneous flow of liquid medium to all parts of said support.
It is another object of the invention to provide a method for the culturing of liver cells, with which liver cells can be kept viable in an amount and during a period of time that are practical for use in a bio-artificial liver.
A further objection of the invention is to provide a bio-artificial liver with improved therapeutic characteristics that can be used to replace and/or supplement the liver function of a patient.
Yet another object of the invention is to provide a method for the treatment of liver failure, especially acute liver failure, by using a bio-artificial liver.
Further objects of the invention will become clear from the description hereinbelow.